1. New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in droplet-based microfluidic applications
- Author
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Estelle Mayot, Gabrielle Woronoff, Oliver J. Miller, Patrice Soumillion, Abdeslam El Harrak, Olivier Schicke, Michael Ryckelynck, and Andrew D. Griffiths
- Subjects
Mesylates ,Models, Molecular ,Fluorophore ,Chromatography ,Molecular Structure ,Microfluidics ,technology, industry, and agriculture ,Leaving group ,Substrate (chemistry) ,Microfluidic Analytical Techniques ,complex mixtures ,Fluorescence ,Analytical Chemistry ,Amidase ,Substrate Specificity ,chemistry.chemical_compound ,Microtiter plate ,Kinetics ,Sulfonate ,chemistry ,Coumarins ,Escherichia coli ,Penicillin Amidase ,Enzyme Assays ,Fluorescent Dyes - Abstract
Droplet-based microfluidics is a powerful tool for biology and chemistry as it allows the production and the manipulation of picoliter-size droplets acting as individual reactors. In this format, high-sensitivity assays are typically based on fluorescence, so fluorophore exchange between droplets must be avoided. Fluorogenic substrates based on the coumarin leaving group are widely used to measure a variety of enzymatic activities, but their application in droplet-based microfluidic systems is severely impaired by the fast transport of the fluorescent product between compartments. Here we report the synthesis of new amidase fluorogenic substrates based on 7-aminocoumarin-4-methanesulfonic acid (ACMS), a highly water-soluble dye, and their suitability for droplet-based microfluidics applications. Both substrate and product had the required spectral characteristics and remained confined in droplets from hours to days. As a model experiment, a phenylacetylated ACMS was synthesized and used as a fluorogenic substrate of Escherichia coli penicillin G acylase. Kinetic parameters (k(cat) and K(M)) measured in bulk and in droplets on-chip were very similar, demonstrating the suitability of this synthesis strategy to produce a variety of ACMS-based substrates for assaying amidase activities both in microtiter plate and droplet-based microfluidic formats.
- Published
- 2011